Novel composition of matter for delivering lipid-soluble materials, and a method for producing it

ABSTRACT

This invention describes a novel composition of matter describing a complex comprising leaf protein and a lipophilic substance(s), along with the method of producing it. Delivery of lipid-soluble materials into the body is challenging because they are generally highly insoluble in water and very subject to oxidative degradation. The inventors have found that leaf protein—the water-soluble proteins derived from plant leaves—can efficiently form a complex with lipophilic materials. This leaf protein-lipid-soluble material complex is an effective carrier of lipophilic substances. As such, the leaf protein-lipid-soluble material complex disclosed herein can be used for the delivery of lipophilic vitamins, fatty acids, caretenoids, lipophilic drugs, and other lipophilic materials. This complex can be used to deliver lipophiles in foods, nutritional and dietary supplements, topical compositions and in pharmaceutical products.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from the provisional application Ser.No. 61/311,072 which was filed Mar. 5, 2010, the entire contents ofwhich are incorporated herein by reference.

GOVERNMENT INTERESTS

This invention was made with U.S. government support under USDA-CSREESAwards Nos. 2008-34467-19445 and 2009-34467-20151. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

For purposes of this invention disclosure, the terms “lipid-soluble” and“lipophilic” refer to compounds or substances which are capable ofdissolving in fats, oils, lipids, or non-polar solvents. The terms“lipid-soluble” and “lipophilic” are used interchangeably, and the term“lipophile” refers to a substance which is lipophilic.

Delivery of lipid-soluble materials such as vitamins A, D, E and K,fatty acids and lipid-soluble pharmaceuticals into the human (or animal)body remains a challenge. It can be difficult to maintain lipid-solublenutrients in low-fat foods because they do not remain in solution and/orthey adsorb to packaging materials (Swaisgood et al., 2001). Existingcommercial delivery and fortification strategies revolve aroundemulsification and microencapsulation, both of which have limitations.Emulsification requires product-specific emulsifiers, many of which arenot GRAS (Generally Recognized As Safe). Microencapsulation materials,such as cyclodextrins, are often expensive. In addition, theseapproaches invariably require using a substantial amount of fat ascarriers for lipid-soluble materials.

The need for new carriers for lipid-soluble materials has becomeparticularly apparent, given the recent resurgence of vitamin Ddeficiencies. Vitamin D is associated with bone health, myocardialdevelopment, brain and fetal development and reduced cancer risk. Whilethe needs are evident, the means to incorporate vitamin D remainlimited, at least in part due to the fact that vitamin D is sensitive toacid, oxygen, and light. Fortification of lipid-soluble vitamins, suchas vitamin D, is challenging given their sensitive chemical nature. Thepresence of conjugated double bonds in vitamin D provides an easy routefor decomposition by oxidation. Isomerization can occur under acidic orlight conditions. Temperatures above 40° C. and relative humidity above85% can deteriorate it, while mild acidification can isomerizes it toinactive forms.

Similarly, fortification of foods and beverages with fatty acids, suchas polyunsaturated Ω-3 fatty acids, is very challenging because thefatty acids are highly insoluble in water and very sensitive tooxidative degradation which can reduce their health benefits and causeundesirable odors (Zimet et al., 2009).

Protein-based carriers offer a potential alternative to existingcarriers, although the limited research to date on protein carriers hasfocused on dairy proteins. Wang et al. (1997) reported thatbeta-lactoglobulin, the major protein in whey, showed substantiallygreater binding affinity to vitamin D₂ than to vitamin A. They did not,however, report being able to produce a complex using beta-lactoglobulinand a vitamin. They also did not provide binding efficiency data whichwould indicate what proportion of the available vitamins the protein wasable to bind.

Swaisgood et al. (2001) also used beta-lactoglobulin to form a complexwith vitamin D. While they were able to form a complex which was solublein aqueous solution, their approach involved affinity purificationmethods, including use of affinity chromatography in their preferredmethod, which would be cost-prohibitive for commercial applications. Theauthors also did not provide information about the proportion of theadded vitamin D which was retained in the complex along withbeta-lactoglobulin.

Zimet et al. (2009) noted that certain food proteins, particularly milkproteins, had an ability to bind to hydrophobic molecules, making themuseful for the encapsulation and delivery of bioactive compounds. Theyreported that beta-lactoglobulin had been found to bind with vitamin D,retinoic acid, cholesterol and various aromatic compounds and fattyacids. They noted, though, that there had been no prior published workon the binding of proteins to Ω-3 fatty acids. Using a complexcontaining beta-lactoglobulin and pectin, they reported an encapsulationefficiency for DHA (docosahexaenoic acid) of approximately 64% (i.e.,amount of DHA encapsulated as a percent of the initially added DHA).

Semo et al. (2007) attempted to use microencapsulation involving purecasein micelles. They wrote that the use of casein micelles as carriersfor nutraceuticals had not yet been reported in the literature. However,they were only able to encapsulate approximately 27% of the analyticallyrecovered vitamin D₂ which they had added to a suspension containingcasein micelles.

The inventors of the present invention have unexpectedly found thatusing a plant-based protein, leaf protein, they were able to create aleaf protein-vitamin D complex which retained approximately 85% of thevitamin D contained in a mixture—more than three times greaterpercentages of vitamin D than is reported from casein micelles. Thepresent invention pertains specifically to the use of leaf proteins in acomplex with lipid-soluble materials.

The term “leaf protein” as used in this invention disclosure is intendedto refer to all water-soluble proteins contained in plant leaves. Theleaf protein may be obtained from any green leafy plant, as it is wellknown that all chlorophyll-containing plants contain soluble leafproteins. Examples of such plants include, but are not limited to,tobacco, alfalfa and spinach. Lo et al. (2008) and Fu et al. (2010) havedescribed a method for efficiently recovering and preparing a leafprotein powder from the leaves of green plants. Leaf protein may beextracted from plants, and a suitable leaf protein powder prepared,using the method described in Lo et al. (2008), which is incorporated byreference, or using other methods which may be known to practitioners ofthe art.

Leaf proteins—the proteins which occur naturally in the leaves of greenplants—are perhaps the most abundant proteins in nature. They containexcellent binding, gelling, foaming, whipping and emulsifyingcharacteristics, and have nutritional value comparable to milk protein(Lo et al., 2008; Sheen et al., 1991). Leaf protein carriers also offeranother advantage over other proteins in that consumers do not have toworry about whether the products contain animal-origin or dairy-basedingredients. Leaf protein is therefore a very desirable carrier for thedelivery of lipophilic substances.

SUMMARY OF THE INVENTION

This invention provides a novel composition of matter comprising acomplex of leaf protein and one or more lipid-soluble materials. Thepresent invention also provides methods of making and using suchcomplexes.

Complexes of the invention typically comprise leaf protein and one ormore lipid-soluble materials such as, for example, vitamins A, D, E, andK, fatty acids, lipid-soluble pharmaceuticals, or other lipid-solublematerials. In a preferred embodiment, the complex is a powdery solidmaterial. This complex is useful as a carrier for the delivery oflipid-soluble materials, into or onto humans or animals. A non-limitinglist of examples for the possible uses of this leafprotein-lipid-soluble material complex are as a food or in food as adelivery system for vitamins or other lipid-soluble materials; indietary supplements and nutraceuticals, infant formulas, inpharmaceuticals or in topical compositions. Nutrient and vitaminsupplements can be in any form known in the art, including but notlimited to, powders, tablets (chewable or otherwise), capsules,gel-caps, elixirs, and effervescent tablets. Alternatively, nutrient andvitamin supplements can be in the form of bars, drinks, juices orshakes, among others. These and other aspects of the present inventionare disclosed in more detail in the description of the invention below.

The inventors were able to produce a leaf protein-vitamin D₃ complexwhich retained approximately 85% of the vitamin D added to a mixture,using a preferred embodiment of the claimed method. (See FIG. 1). Thisresult indicates that leaf protein is highly effective and efficient asa carrier of lipid-soluble materials. Without wishing to be bound bytheory, this result also indicates that leaf protein has many bindingsites, and is able to carry large amounts of target lipid-solublematerials.

Methods of the invention typically comprise preparing a suspensioncontaining leaf protein. Optionally, non-water-soluble materials may beremoved from the leaf protein suspension. Lipid-soluble materials maythen be mixed with the suspension. Optionally, lipid-soluble materialsmay be prepared by dissolving them in a solvent prior to their additionto the suspension. When the leaf protein suspension and thelipid-soluble materials have been suitably combined, they may be furthertreated. In one embodiment, the mixture may be frozen and, optionally,lyophilized. In some embodiments, the mixture may be dried withoutfreezing using techniques well known in the art, for example, spraydrying. In some embodiments, the mixture is dried into a powder. Theresulting product is a solid powder containing a complex of leaf proteinand the target lipid-soluble material(s).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Comparison of vitamin D₃ recovery in freeze-dried formulation:with tobacco leaf protein vs. control.

FIG. 2: Comparison of vitamin D₃ recovery, and crude protein % invitamin D-tobacco leaf protein complex of different water content.

FIG. 3: Comparison of vitamin D₃ recovery, and crude protein % invitamin D-tobacco leaf protein complex of varying pH.

FIG. 4: Solubility values of vitamin D-tobacco leaf protein complexformulated at different pH (p value=0.0709).

FIG. 5: Comparison of vitamin D₃ recovery and crude protein % in thevitamin D-tobacco leaf protein complex under different mixingconditions.

DETAILED DESCRIPTION OF INVENTION

The principles, preferred embodiments and modes of operation of thepresent invention will be described hereunder. The invention which isintended to be protected herein should not, however, be construed aslimited to the particular forms disclosed, as these are to be regardedas illustrative rather than restrictive. Variations and changes may bemade by those skilled in the art without departing from the spirit ofthe present invention. Accordingly, the examples, descriptions, and bestmode of carrying out the invention given below should be consideredexemplary in nature and not as limiting to the scope and spirit of theinvention as set forth in the claims.

The objective of the methods of this invention is to produce a complexcontaining leaf protein and one or more lipid-soluble substances. Thepresent invention may be used to prepare complexes consisting of leafprotein and any other lipid-soluble materials, including but not limitedto, vitamins D₃, A, E, K, other types of vitamin D, fatty acids such asDHA, eicosapentaenoic acid, linoleic acid, and alpha-linoleic acid,lipid-soluble drugs (some of which are listed below), cholesterol,retinol and retinoids and other lipophilic substances. In someembodiments, complexes of the invention may comprise 2, 3, 4, 5, or morelipid-soluble materials.

The term “target substance” as used in this invention disclosure refersto the particular lipid-soluble substance(s) which the practitionerwishes to form into a complex with leaf protein.

The present invention is based on the discovery that leaf protein veryefficiently forms complexes with lipophilic substances, for examplevitamin D. This property allows the use of leaf protein as a carrier forlipophilic nutrients in foods, dietary supplements and nutraceuticals,infant formulas, drugs and pharmaceuticals and topical compositions.

Lipid-soluble materials may be derived from any source known in the art,for example, the vitamin A, vitamin D, vitamin E, and vitamin K as usedherein can be from any source known in the art. The term “vitamin A” asused herein refers to any form of vitamin A, including but not limitedto, retinol, retinaldehydes, retinal, retinoic acid (also known astretinoin and retin-A), and vitamin A salts and derivatives (e.g.,retinol palmitate, retinyl acetate, and β-carotene and othercarotenoids). The term “vitamin D” as used herein refers to any form ofvitamin D, including but not limited to, ergocalciferol (D₂),cholecalciferol (D₃), 22,23-dihydroergocalciferol (D₄), and vitamin Dsalts and derivatives (e.g., 25-hydroxycholecalciferol and1-α,25-dihydroxycholecalciferol). The term “vitamin E” as used hereinrefers to the family of compounds known as tocopherols (e.g.,α-tocopherol, β-tocopherol, δ-tocopherol, γ-tocopherol), as well astocol, tocoquinone, tocotrienol, and vitamin E salts (e.g., vitamin Ephosphate) and derivatives (e.g., tocopherol sorbate, tocopherolacetate, tocopherol succinate, other tocopherol esters). As used herein,the term “vitamin K” refers to vitamin K₁ (phytonadione), vitamin K₂(menaquinone), vitamin K₃ (menadione), vitamin K₄, vitamin K₅, vitaminK₆, vitamin K₇, and their salts and derivatives.

Fatty acids refer to carboxylic acids with a long unbranched aliphatictail, and which are either saturated or unsaturated. Fatty acidsinclude, but are not limited to DHA, eicosapentaenoic acid, linoleicacid, and alpha-linoleic acid, amongst many others.

Leaf protein powder suitable for practicing this invention may beobtained from plant leaves using the method described in Lo et al.(2008) and Fu et al. (2010), or using any other method of leaf proteinprocessing or extraction which may be known to practitioners in the art.

One suitable method for preparing leaf protein is as follows:

Freshly harvested green plant leaves may be chopped with a hammermill.The leaves can be either freshly harvested, or they can be stored in acool or frozen state or dried following harvest until they are ready forprocessing. Alternatively, physical maceration procedures, combined withmechanical pressure, can be utilized to disrupt the cell wall andprepare the proteins for solubilization.

Substantially simultaneously with the leaf rupturing, a buffer solutionis added to the leaves. The inventors found that a solution containingsodium phosphate dibasic and potassium phosphate monobasic(Na₂HPO₄—KH₂PO₄) is especially effective, although other effectivebuffering agents may be used. The inventors also found that a pH of 7.77is preferable as it gave the highest protein yields with this agent,although a pH range between approximately 7.4-8.0 or even 6.5-9.0 isacceptable.

It is preferred that the buffer should have a low concentration, inorder to avoid precipitating or denaturing the proteins. It was foundthat a buffer concentration of approximately 0.067M was the optimalconcentration, although a range of 0.025M to about 0.3M is quiteacceptable, and more preferably a range of about 0.067M-about 0.2 M.

It is preferred that the buffer solution should also contain both achelating agent and a reducing agent. The purpose of the chelating agentis to remove loose ions from the resulting juice. We have found that 10mM of EDTA, a well-known chelating agent, is effective to recover looseions. The purpose of the reducing agent is to prevent oxidation anddenaturation of the proteins. We have also found that 25 mM of2-mercaptoethanol is effective as a reducing agent.

The ruptured leaves may be stored in the buffer solution for up totwenty-four hours, although preferably not more than five hours. Whilesuch storage is not necessary, it was found to help improve ultimateprotein recovery.

An industrial filter may then be used to filter out the fibrous leafbiomass, leaving a green juice containing the soluble protein. We havefound a screw press to give effective results. This green juice containsthe soluble proteins along with plant chloroplast materials. Subjectingthis green juice to powerful centrifuge will remove this chloroplastmaterial, leaving an “amber juice” which contains the soluble proteins.Centrifuging at a force of approximately 12,000 g for approximately 20minutes is sufficient to remove leaf chloroplast materials. Eithercontinuous centrifuge or disk centrifuge is suitable. However, failureto adequately centrifuge the green juice will result in incompleteremoval of the chloroplasts, and can leave an undesirable green tint inthe resulting proteins.

Depending upon the desired use, it is possible to obtain severaldifferent protein products from this amber juice.

Product 1—Crude Protein Powder. The simplest approach is to prepare aprotein powder product from this resulting amber juice through the useof standard industrial drying processes. This powder product can beprepared using spray drying, vacuum drying or freeze drying. However,spray drying is most practical for scale-up to an industrial level. Thiscrude protein powder could be satisfactory for many commercial uses.

Product 2—Purified Protein Powder. It is possible to remove nucleicacids and small molecule impurities through a precipitation of the amberjuice solution at its isoelectric point, which we have found to be at orabout pH 5.3 (±0.5). The resulting solution can then be dried via spraydrying or other industrial drying techniques to obtain a more highlypurified powder product.

Product 3—It is possible to separate ribulose 1.5-bisphosphatecarboxylase/oxygenase (RuBisCO) from the amber juice. An isoelectricpoint precipitation can be conducted at a pH of approximately 5.3(±0.5), which is the isoelectric point for RuBisCO. This protein canthen be centrifuged at a force of approximately 12,000 g or greater. Theprecipitate is then resuspended in buffer solution at a pH ofapproximately 7.77. The precipitate is then dried using spray drying orother means to produce a powder product containing RuBisCO. RuBisCO canbe further purified if desired.

Product 4—The supernatant from the isoelectric point precipitation at pH5.3 can be further purified to yield other leaf proteins. A secondisoelectric point precipitation can be conducted involving thesupernatant at a pH of approximately 4.2 (±0.5). The proteins can thenbe resuspended in buffer at a pH of approximately 7.77, and then driedusing spray drying or other forms of drying.

Any of the above-described leaf protein powders may be used in thepractice of the present invention.

The leaf protein powder is then placed in a suitable solvent, such aswater, to form a suspension. Failure to place the protein powder into asuitable solvent may inhibit or prevent formation of an effectiveprotein complex, as the dry protein is generally too coarse toefficiently bind or form a complex with a target substance.Additionally, preparing this protein-containing suspension is believedto expose additional binding sites to the target substance. In apreferred embodiment, water is used as the solvent to form the leafprotein aqueous suspension. In a particularly preferred embodiment, theratio of leaf protein powder to water will be approximately 1 gram ofleaf protein powder to between approximately 30 to 80 ml of water.Failure to maintain adequate water content will reduce the capacity ofleaf protein to form a complex with the target substance. Use ofexcessive water content may add drying time and cost, and may reduceinteraction of the protein and target substance.

In a preferred embodiment, the pH of the water is adjusted to between3.3 and 6.3. Using a pH below this range may degrade the leaf protein.Using a pH in the preferred range maintains the structure of theprotein, which optimizes its ability to retain the target substance.

In the present invention, the leaf protein forms a complex with one ormore target substances in solution. The target substance(s), which is alipid-soluble material, is solubilized in a suitable organic solvent,such as ethanol, methanol, other alcohols, hexane, acetone, or toluene,amongst many others. One skilled in the art will realize that theoptimal organic solvent will depend on the nature of the lipid-solublematerial. As an example, ethanol is particularly preferred if vitamin D₃is the target substance.

Following solubilization of the target substance(s), theprotein-containing suspension and the target-substance(s)-containingsolution are then mixed together and then, in a preferred embodiment,frozen. Several techniques known to practitioners in the art mayoptionally be used to enhance mixing, for example, magnetic stirring,sonication, vortexing, or a combination of mixing methods. Practitionersin the art will recognize that different mixing techniques may provemore suitable for particular lipid-soluble materials than othertechniques. If vitamin D₃ is the target substance, a preferredembodiment is the use of magnetic stirring for approximately fiveminutes.

In a preferred embodiment, freezing should occur within one hour afterthe protein-containing suspension is mixed with thetarget-substance-containing solution. In a more preferred embodiment,freezing occurs substantially immediately after the protein-containingsuspension is mixed with the target-substance-containing solution.Delays in freezing after mixing the protein-containing suspension withthe target-substance-containing solution may reduce the amount of thetarget substance which forms a complex with the leaf protein. At thetime of mixture, the protein and target substance are in close contact.However, they may separate as time is allowed to pass. One skilled inthe art will recognize that the rate at which the protein and targetsubstance dissociate will depend on the nature of the targetsubstance(s), and that this will affect the optimal time for freezing tooccur.

Any techniques which obtain substantially immediate freezing of theprotein-target substance mixture are potentially suitable. Anon-limiting list of suitable freezing techniques include use of liquidnitrogen, dry ice or methanol. A preferred embodiment is the use ofliquid nitrogen for freezing.

In a preferred embodiment, following freezing, the frozen mixture ofprotein and the target substance(s) is then dried. Any technique fordrying may be suitable, including but not limited to freeze-drying,precipitation, oven-drying, microwaving or a combination of methods. Onepreferred embodiment is freeze-drying, as this technique will notdegrade the protein or target substance. If freeze-drying is used, thenthe end product will be a powdery material containing a complex whichcontains leaf protein and the target substance.

The resulting dried complex, containing leaf protein and thelipid-soluble target substance(s), is suitable for use as a foodadditive, in forming nutrient, vitamin or other dietary supplements ornutraceuticals. Such products can be in any form known in the art,including but not limited to, tablets (including chewable tablets),capsules, gel-caps, powders, elixirs, and effervescent tablets.Alternatively, such products can be in the form of shakes, juices orother drinks, and bars.

The present invention also provides food compositions comprisingcomplexes of leaf protein and lipophilic nutrients. Preferably thelipophilic nutrients are vitamin A, vitamin D, vitamin E, vitamin K₁,cholesterol, carotenoids, conjugated linoleic acid, essential fattyacids, and other fatty acids. Because of its excellent nutritionalqualities and water-solubility, leaf protein is highly suitable as asuitable carrier for lipophilic nutrients in food compositions.Complexes of leaf proteins and lipophilic nutrients are also useful forfortifying infant formulas with DHA and other lipid-soluble substances.

The food compositions of the present invention are formed by combining aleaf protein-lipid-soluble material complex according to the presentinvention with other food ingredients. Alternately stated, a foodcomposition is a food product containing a leaf protein-lipid-solublematerial complex of the present invention as an ingredient or component.A food composition can be a liquid or a solid food for human or animalconsumption, and includes, but is not limited to, dairy products,processed meats, breads, cakes and other bakery products, processedfruits and vegetables, etc.

The present invention also includes compositions comprising a leafprotein-lipid soluble material complex, in which the leaf protein formsa complex with a lipophilic drug for delivery into humans or animals.Such compositions can be in any form known in the art, including but notlimited to, tablets (including chewable tablets), capsules, gel-caps,powders, elixirs, and effervescent tablets. A non-limiting list oflipophilic drug substances which may used to form aleaf-protein-lipid-soluble material complex according to the presentinvention includes the following: Analgesics and anti-inflammatoryagents: aloxiprin, auranofin, azapropazone, benorylate, diflunisal,etodolac, fenbufen, fenoprofen calcim, flurbiprofen, ibuprofen,indomethacin, ketoprofen, meclofenamic acid, mefenamic acid, nabumetone,naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac;Anthelmintics: albendazole, bephenium hydroxynaphthoate, cambendazole,dichlorophen, ivermectin, mebendazole, oxamniquine, oxfendazole, oxantelembonate, praziquantel, pyrantel embonate, thiabendazole;Anti-arrhythmic agents: amiodarone, disopyramide, flecamide acetate,quinidine sulphate; Anti-bacterial agents: benethamine penicillin,cinoxacin, ciprofloxacin, clarithromycin, clofazimine, cloxacillin,demeclocycline, doxycycline, erythromycin, ethionamide, imipenem,nalidixic acid, nitrofurantoin, rifampicin, spiramycin, sulphabenzamide,sulphadoxine, sulphamerazine, sulphacetamide, sulphadiazine,sulphafurazole, sulphamethoxazole, sulphapyridine, tetracycline,trimethoprim; Anti-coagulants: dicoumarol, dipyridamole, nicoumalone,phenindione; Anti-depressants: amoxapine, maprotiline, mianserin,nortriptyline, trazodone, trimipramine maleate; Anti-diabetics:acetohexamide, chlorpropamide, glibenclamide, gliclazide, glipizide,tolazamide, tolbutamide; Anti-epileptics: beclamide, carbamazepine,clonazepam, ethotoin, methoin, methsuximide, methylphenobarbitone,oxcarbazepine, paramethadione, phenacemide, phenobarbitone, phenyloin,phensuximide, primidone, sulthiame, valproic acid; Anti-fungal agents:amphotericin, butoconazole nitrate, clotrimazole, econazole nitrate,fluconazole, flucytosine, griseofulvin, itraconazole, ketoconazole,miconazole, natamycin, nystatin, sulconazole nitrate, terbinafine,terconazole, tioconazole, undecenoic acid; Anti-gout agents:allopurinol, probenecid, sulphin-pyrazone;] Anti-hypertensive agents:amlodipine, benidipine, darodipine, dilitazem, diazoxide, felodipine,guanabenz acetate, isradipine, minoxidil, nicardipine, nifedipine,nimodipine, phenoxybenzamine, prazosin, reserpine, terazosin;Anti-malarials: amodiaquine, chloroquine, chlorproguanil, halofantrine,mefloquine, proguanil, pyrimethamine, quinine sulphate; Anti-migraineagents: dihydroergotamine mesylate, ergotamine tartrate, methysergidemaleate, pizotifen maleate, sumatriptan succinate; Anti-muscarinicagents: atropine, benzhexyl, biperiden, ethopropazine, hyoscyamine,mepenzolate bromide, oxyphencylcimine, tropicamide; Anti-neoplasticagents and Immunosuppressants: aminoglutethimide, amsacrine,azathioprine, busulphan, chlorambucil, cyclosporin, dacarbazine,estramustine, etoposide, lomustine, melphalan, mercaptopurine,methotrexate, mitomycin, mitotane, mitozantrone, procarbazine, tamoxifencitrate, testolactone. tacrolimus, sirolimus; Anti-protozoal agents:benznidazole, clioquinol, decoquinate, diiodohydroxyquinoline,diloxanide furoate, dinitolmide, furzolidone, metronidazole, nimorazole,nitrofurazone, omidazole, timidazole; Anti-thyroid agents: carbimazole,propylthiouracil; Alixiolytic, sedatives, hypnotics and neuroleptics:alprazolam, amylobarbitone, barbitone, bentazepam, bromazepam,bromperidol, brotizolam, butobarbitone, carbromal, chiordiazepoxide,chlormethiazole, chlorpromazine, clobazam, clotiazepam, clozapine,diazepam, droperidol, ethinamate, flunanisone, flunitrazepam,fluopromazine, flupenthixol decanoate, fluphenazine decanoate,flurazepam, baloperidol, lorazepam, lormetazepam, medazepam,meprobamate, methaqualone, midazolam, nitrazepam, oxazepam,pentobarbitone, perphenazine pimozide, prochlorperazine, sulpiride,temazepam, thioridazine, triazolam, zopiclone; beta-Blockers:acebutolol, alprenolol, atenolol, labetalol, metoprolol, nadolol,oxprenolol, pindolol, propranolol; Cardiac Inotropic agents: amrinone,digitoxin, digoxin, enoximone, lanatoside C, medigoxin; Corticosteroids:beclomethasone, betamethasone, budesonide, cortisone acetate,desoxymethasone, dexamethasone, fludrocortisone acetate, flunisolide,flucortolone, fluticasone propionate, hydrocortisone,methylprednisolone, prednisolone, prednisone, triamcinolone; Diuretics:acetazolamide, amiloride, bendrofluazide, bumetanide, chlorothiazide,chlorthalidone, ethacrynic acid, frusemide, metolazone, spironolactone,triamterene; Anti-parkinsonian agents: bromocriptine mesylate, lysuridemaleate; Gastro-intestinal agents: bisacodyl, cimetidine, cisapride,diphenoxylate, domperidone, famotidine, loperamide, mesalazine,nizatidine, omeprazole, ondansetron, ranitidine, sulphasalazine;Histamine H-Receptor Antagonists: acrivastine, astemizole, cinnarizine,cyclizine, cyproheptadine, dimenhydrinate, flunarizine, loratadine,meclozine, oxatomide, terfenadine; Lipid regulating agents: bezafibrate,clofibrate, fenofibrate, gemfibrozil, probucol; Nitrates and otheranti-anginal agents: amyl nitrate, glyceryl trinitrate, isosorbidedinitrate, isosorbide mononitrate, pentaerythritol tetranitrate; HIVprotease inhibitors: Nelfinavir; Opioid analgesics: codeine,dextropropyoxyphene, diamorphine, dihydrocodeine, meptazinol, methadone,morphine, nalbuphine, pentazocine; Sex hormones: clomiphene citrate,danazol, ethinyl estradiol, medroxyprogesterone acetate, mestranol,methyltestosterone, norethisterone, norgestrel, estradiol, conjugatedoestrogens, progesterone, stanozolol, stibestrol, testosterone,tibolone; Stimulants: amphetamine, dexamphetamine, dexfenfluramine,fenfluramine, and mazindol. (See Benita et al., 2007 regarding a list oflipophilic drugs).

The present invention also includes compositions for use in personalcare and/or hygiene comprising the leaf protein-lipid-soluble materialcomplexes disclosed herein (e.g., soaps, skin creams, soaps, cleansers,shampoos). Topical compositions containing complexes of leaf proteinwith vitamin E, vitamin A, conjugated linoleic acid, and essential fattyacids are preferred. The topical compositions disclosed herein aresuitable for topical application to mammalian skin. The compositionscomprise a safe and effective amount of the leaf protein complexes andother active agents, and a cosmetically- and/orpharmaceutically-acceptable topical carrier.

The phrase “cosmetically- and/or pharmaceutically-acceptable carrier”,as used herein, means any substantially non-toxic carrier suitable fortopical administration to the skin, which generally has good aestheticproperties, and is compatible with the leaf protein-lipid-solublematerial complexes of the present invention. By “compatible” it is meantthat the leaf protein-lipid-soluble material complexes will remainstable and retain substantial activity therein. The carrier can be in awide variety of forms, such as sprays, emulsions, mousses, liquids,creams, oils, lotions, ointments, gels and solids. Suitablepharmaceutically-acceptable topical carriers include, but are notlimited to, water, glycerol, alcohol, propylene glycol, fatty alcohols,triglycerides, fatty acid esters, and mineral oils. Suitable topicalcosmetically-acceptable carriers include, but are not limited to, water,petroleum jelly, petrolatum, mineral oil, vegetable oil, animal oil,organic and inorganic waxes, such as microcrystalline, paraffin andozocerite wax, natural polymers, such as xanthanes, gelatin, cellulose,collagen, starch or gum arabic, synthetic polymers, alcohols, polyols,and the like. Preferably, because of its non-toxic topical properties,the pharmaceutically- and/or cosmetically-acceptable carrier issubstantially miscible in water. Such water miscible carriercompositions can also include sustained or delayed release carriers,such as liposomes, microsponges, microspheres or microcapsules, aqueousbased ointments, water-in-oil or oil-in-water emulsions, gels and thelike.

The disclosed complex is also suitable as a component of tissue culturemedia or microbial growth media to promote growth, differentiationand/or viability of cultured cells. Milk proteins have been shown to bea suitable fatty acid carrier in cell culture (Swaisgood et al., 2001),and therefore leaf proteins should be similarly suitable.

EXAMPLES Example 1

Evaluation of Different Strategies for Solubilizing Leaf Protein.

The purpose of this test was to evaluate different strategies forsolubilizing the leaf protein. It is necessary to solubilize the leafprotein in order to remove residual pigments, fat content and other forma complex with the target lipid-soluble molecules.

Protein samples were subjected to solvent extraction with three organicsolvents; hexane; acetone and methanol. Leaf protein powder prepared bythe method of Lo et al. (2008) and Fu et al. (2010) has a watersolubility value of 10.08±0.15 grams/liter (g/l). Hexane extraction ofthe protein powder yielded only a marginal increase in solubility of10.82 g/l, whereas acetone extraction showed even smaller increase insolubility and methanol actually caused solubility to decrease. Based onthese findings, the inventors did not utilize a solvent as pretreatmentprior to mixing with vitamin D.

Example 2

Effect of Water Content on Leaf Protein-Lipophile Complex

Leaf protein samples were derived from Maryland tobacco variety 609LA, alow-alkaloid variety containing 0.6 mg/g to 0.8 mg/g of nicotine, usingthe method described in Lo, et al. (2008) and Fu et al. (2010). One gramof leaf protein powder was placed in a 300-ml freeze-drying glass flask(F05657000, Thermoscientific, Pittsburgh, Pa.), followed by addition ofeither 20 ml or 40 ml of water and 4 ml of vitamin D₃ in 99% pureethanol (1000 ug/ml). The pH of the mixture was adjusted to pH 4.3 bygradually adding 1 M sodium hydroxide solution to the protein watersolution prior to adding vitamin D₃. This mixture was then magneticallystirred for 4 minutes before liquid nitrogen was added. Approximately250 ml of liquid nitrogen was poured into the glass until the mixtureappeared completely solid. The flask was then immediately closed withthe lid and carefully placed in a thermally insulated bag filled withdry ice. The connector end of the freeze-drying flask was connected tothe freeze-dryer (RVT4104 model Refrigerated Vapor Trap, Thermo ElectronCorporation, NY) at −110° for 96 hours.

Using 40 ml of water per gram of leaf protein powder to obtain thevitamin D-protein complex, the inventors obtained a vitamin D₃ recoveryof 84.68±3.92% of the total vitamin D₃ added. In contrast, use of only20 ml of water per gram of protein powder significantly reduced thevitamin D₃ recovery to 70.21±8.92%. (See FIG. 2). The inventors observedthat the spherical structure in the protein aggregates could not bemaintained at the lower water content levels. Without wishing to bebound by theory, the inventors hypothesize that the increased waterlevel at 40 ml helped form hydrogen bonds which maintained the proteinstructure. Conversely, at the lower water content level (whichcorresponded with higher protein density), self-stabilization ofproteins may have taken place where proteins tended to form bonds whichinterconnected adjacent proteins, reducing the sites available forvitamin D₃ binding as well as limited surface area of ice-/waterinterface during the freeze-drying process.

Example 3

Effect of pH on Leaf Protein—Lipophile Complex

The inventors measured the effect of pH on the leaf protein—lipophilecomplex. They used the same preparation as described above in Example 2,except that they only used one water content level: one gram of leafprotein per 40 ml of water. They also prepared the leaf protein-vitaminD mixture described in Example 2 at three different pH levels (4.3, 8.5and 11.0). As noted above, for pH adjustment, 1 M sodium hydroxidesolution was gradually added to the protein water solution prior toadding vitamin D₃.

The sample tested at pH 4.3 showed substantially higher recovery ofvitamin D₃ (84.6%±3.92%, w/w) than either of the two other treatments.(See FIG. 3). There was also a slight increase in the water solubilityof the vitamin D-protein complex at pH 4.3 from 10.08 to 10.78 g/l (SeeFIG. 4).

In each of the three treatments, the crude protein represented about 30%of the vitamin D₃-tobacco leaf protein complex.

It is generally recognized that changes in pH can induce significantalterations in protein structure. At pH 4.3, the vitamin _(D3)-proteincomplex appeared to be spherical aggregates. As the pH increased to 8.5,the spherical structure opened up and bridged with adjacent aggregates,forming an interwoven structure. At pH 11.0, the spherical structure wascompletely disrupted, forming a continuous porous structure. In otherwords, porosity increased as the pH increased, corresponding to the lossof vitamin D. Without wishing to be bound by theory, the inventorsbelieve that this increase in porosity was related to a loss of vitaminD. Again without wishing to be bound by theory, the inventors believethat that the lower porosity and spherical aggregate structure of theprotein at the lower pH permitted the protein to retain or “trap” thevitamin D so it could not escape.

Example 4

Effect of Protein-Vitamin Mixing Technique on Vitamin Carrying Capacity

This test measured the effect of different techniques for mixing proteinand vitamin D₃ on the protein's vitamin D₃ carrying capacity. They usedthe same preparation as described above in Example 2, except that theyonly used one water content level: one gram of leaf protein per 40 ml ofwater. The inventors also tested three mixing treatments: (i) magneticstirring for 5 minutes, (ii) a Sonicator (28H ultrasonic bath, Neytech,Bloomfield, Conn.) at a frequency of 47±3 khZ for 5 minutes; and (iii) acombination involving the sonication treatment followed by the stirringtreatment.

The highest vitamin recovery was obtained when the samples werestir-mixed for 5 minutes, reaching 84.68%±3.92%, w/w (weight/weight).Sonication alone resulted in a substantial reduction in vitamin D₃recovery 62.53%±3.68%, w/w. The temperature increased by 20° C.following sonication. Without wishing to be bound by theory, theinventors hypothesize that the sharp change in temperature may havecaused degradation of vitamin D₃. Vitamin D₃ recovery was lowest whenthe samples were treated by both sonication and mixing (56.32±5.11%,w/w), likely due to the exposure of vitamin D₃ under elevatedtemperature for an extension of 5 minutes during the mixing process.(See FIG. 5). The crude protein content remained statistically the samein all three differentially mixed formulations.

REFERENCES

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All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

Modifications may be made without departing from the basic spirit of thepresent invention. Accordingly, it will be appreciated by those skilledin the art that within the scope of the appended claims, the inventionmay be practiced other than has been specifically described herein.

1-12. (canceled)
 13. A method of preparing a leaf protein-lipid solublematerial complex comprising the following steps: (a) preparing asuspension containing leaf protein in a suitable solvent; and(b)combining the leaf protein suspension with the lipid-solublematerial.
 14. The method of claim 13 wherein non-water-soluble materialis removed from the leaf protein after it is put into suspension. 15.The method of claim 13 wherein the lipid-soluble material is solubilizedin a solvent before it is combined with the leaf protein suspension. 16.The method of claim 13, wherein in (b) the leaf protein in suspension iscombined with lipid-soluble material in a solvent by means of magneticstirring, sonication, manual stirring, shaking, or any combination ofthe foregoing.
 17. The method of claim 13 wherein theleaf-protein-lipid-soluble material mixture is frozen after thecomponents are combined.
 18. The method of claim 17 wherein the mixtureis frozen within one hour of combining the leaf protein and thelipid-soluble material.
 19. The method of claim 17 wherein the freezingis performing by application of liquid nitrogen; dry ice, or storage ata sub-freezing temperature, or a combination of these techniques. 20.The method of claim 17 wherein the mixture is frozen substantiallyimmediately after combining the leaf protein and the lipid-solublematerial.
 21. The method of claim 13 wherein theleaf-protein-lipid-soluble substance(s) mixture is dried to a powderafter it is mixed.
 22. The method of claim 21 wherein the drying isperformed by freeze-drying, precipitation, spray drying, oven-drying,microwaving, or a combination of these techniques.
 23. The method ofclaim 17, wherein the frozen mixture is freeze-dried.
 24. The method ofclaim 13, wherein the solvent in (a) is water.
 25. The method of claim13, wherein the solvent in (b) is an organic solvent.
 26. The method ofclaim 25, wherein the organic solvent comprises ethanol, methanol,another alcohol, hexane, toluene, or acetone.